Background: Platelet glycoprotein VI (GPVI) receives interest when developing new antiplatelet drugs with low bleeding risk. GPVI interactions with collagen initiates thrombus formation and GPVI binding to fibrin(ogen) promotes thrombus growth and stability. To be considered to have the greatest efficacy, GPVI antagonists are expected to inhibit the interaction of GPVI with its main ligands. Glenzocimab is a clinical grade anti-GPVI humanized antibody fragment that inhibits GPVI interaction with collagen and fibrin(ogen).

Aims: To determine the mechanism of the glenzocimab’s inhibitory effect.

Methods: The extracellular domain of GPVI expressed as a monomer (GPVIex) or a dimer (GPVI-Fc) and GPVI-Fc des129-136, were produced and their interaction with glenzocimab analyzed by SPR and solid phase assays. A co-crystal of glenzocimab with GPVIex was obtained and the 1.9 Å structure compared to known GPVI structures (PBB ID: 2GI7 and 5OU7) and GPVI-CRP complex structures (PDB ID: 5OU8 and 5OU9).

Results: Glenzocimab binds to the D2 domain of GPVI. Rearrangements within this domain prevent D2 homotypic interactions and formation of GPVI dimers of high affinity for collagen and fibrin. Glenzocimab induces allosteric modifications within the D1 domain with alterations of the betaC and betaF strands in the CRP binding groove. A shift of the betaC strand results in movement of R38, a key residue into the CRP binding channel and to a direct clash with CRP. Moreover, the light variable region of the GPVI-bound Fab causes steric hindrance which prevents the CRP/collagen elongated chains to extend out of their binding site. Truncation of D2 residues 129-136 blocked binding to glenzocimab, validating the epitope localization.

Conclusion(s): This study demonstrates that altogether, the inhibition of GPVI-dimerization, allosteric modifications of the collagen-binding groove and steric hindrance by glenzocimab allow the inhibition of GPVI interactions with its major ligands.

Figure 1: Glenzocimab inhibits CRP/collagen binding through allosteric modification of the CRP binding groove.

Zoomed in view of the CRP binding groove of the glenzocimab bound and CRP bound -PDB:5OU8- GPVI structures shown in white and green respectively with CRP shown in cyan. The CRP binding groove is largely made from the βC and βF strands within D1. In the glenzocimab bound structure the βC strand is shorter and angled downwards. Due to this movement, R38, a key CRP binding residue, shifts towards CRP and directly clashes with a hydroxyproline residue. The side chain of R38 in both structures is shown as a stick and the shift is shown with a red dotted line. In addition the βF strand in the glenzocimab bound structure is not continuous and forms two smaller strands labelled βF and βF’.

Figure 2: Glenzocimab inhibits CRP/collagen binding through steric hindrance

Surface representation of the glenzocimab bound structure of GPVI with CRP binding superimposed from PDB:5OU8. GPVI, CRP and glenzocimab heavy and light chains are coloured in white, cyan, light pink and light blue respectively. The binding of glenzocimab to the D2 domains means the light variable region of the Fab lies directly in the way of the bound CRP chain. Larger CRP and collagen chains would be blocked from binding due to the presence of the light chain.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/targeting-platelet-glycoprotein-vi-with-glenzocimab-a-novel-mechanism-of-inhibition/